Microarray technologies can facilitate detection of many features per square centimeter. This can include detection via probe binding methodologies to detect or accurately quantify the presence of biomolecules and to characterize these biomolecules, e.g., by determining a specific conformation or sequence. As more information continues to be processed at faster rates, certain features start to become problematic as limiting to the amount of information that can be obtained. For example, many detection technologies, such as probe detection and sequencing rely on monitoring fluorophores and distinguishing fluorophores bound to known probes. The use of fluorophore tags limits the size of the features on a chip due to the diffraction limit, and also can be difficult to detect at small concentrations. Alternative detection technologies exist, but they need further development to provide a suitable improvement to fluorophore-based detection technologies. Therefore, what are needed are alternatives to fluorophore-based detection technologies to improve detection accuracy and facilitate a reduction of feature size for higher throughput and more efficient detection.
As one example, a typical solid support-based detection assay is generally comprised of probes that bind to biologically relevant or active molecules for example, RNA, DNA, or peptides. Probes that bind to target molecules or the target molecules themselves can be covalently attached to a solid planar surface for example, glass, polymer (bead or even plastic composites), or most often, a silicon chip. Additionally, instruments are needed to handle samples (automated robotics), to read the reporter molecules (scanners) and analyze the data (bioinformatic tools). Recently, science has moved toward a unitary machine to perform these much need analyses. In order to marry the chemistry and biology with electronics, silicon wafers are most often used as the solid support or substrate. The term “lab on a chip” has since been coined to describe such an arrangement.
Microarrays technology can facilitate monitoring of many probes per square centimeter. The advantages of using multiple probes include, but are not limited to, speed, adaptability, comprehensiveness and the relatively cheaper cost of high volume manufacturing. The uses of such an array include, but are not limited to, diagnostic microbiology, including the detection and identification of pathogens, investigation of anti-microbial resistance, epidemiological strain typing, investigation of oncogenes, analysis of microbial infections using host genomic expression, and polymorphism profiles.
Recent advances in genomics have culminated in sequencing of entire genomes of several organisms, including humans. Genomics alone, however, cannot provide a complete understanding of cellular processes that are involved in disease, development, and other biological phenomena; because such processes are often directly mediated by polypeptides. Given that huge numbers of polypeptides are encoded by the genome of an organism, the development of high throughput technologies for analyzing polypeptides, amongst many other diverse biomolecules, is of paramount importance.
Peptide arrays with distinct analyte-detecting regions or probes can be assembled on a single substrate by techniques well known to one skilled in the art. A variety of methods are available for creating a peptide microarray. These methods include: (a) chemo selective immobilization methods; and (b) in situ parallel synthesis methods which can be further divided into (1) SPOT synthesis and (2) photolithographic synthesis. These methods are labor intensive and not suited for high throughput. These peptide arrays are expensive to manufacture, have low repeatability, may be unstable, require stringent storage conditions, take a long time to manufacture, and are limited in other ways. Further, while peptide-nucleic acid arrays are useful for identifying biomolecules, there is currently no way to deduce the binding strength or sequence.
What is needed therefore, are improved substrates or arrays and methods to elucidate and replicate biomolecule sequences and measure the binding of one or more biomolecules.
As another specific example, next generation sequencing technologies, including sequencing-by-synthesis, continue to pursue the goal of providing rapid sequencing data at a reasonable cost. This can be used to provide improved health care through individualized medicine and improved diagnostics. Despite many improvements in the past decades, this technology still has limitations in cost and throughput that prevent widespread use. Overcoming these limitations can provide a dramatic impact in several fields, including comparative genomics, high-throughput polymorphism detection, mutation screening, metagenomics, and transcriptome profiling.
Sequencing by synthesis of template DNA bound to a surface is commonly done using fluorophore-labeled, reversible terminator nucleotides. These nucleotides generate a signal corresponding to the sequence of a surface-bound template strand when incorporated into a complementary growing strand. For example, U.S. Pat. No. 7,622,279 teaches a fluorescence-based method for sequencing four modified nucleotides with photo-cleavable fluorescence molecules bound to the side chain of the four nucleic acid bases.
However, optical detection methods have a limited minimum feature size due to diffraction limited detection of fluorophores. Furthermore, imaging of an array of signals and processing the image to generate discrete endpoints can take time and be computationally demanding. Thus, alternative methods of nucleotide identity detection, such as electronic detection are also being explored.
One such method of electronic detection, Ion Sensitive Field Effect Transistors (ISFET), is able to detect small changes in the pH of a reaction volume. Non-optical genome sequencing using ISFET has been performed by adding a single nucleotide at a time to detect the release of an H+ ion upon incorporation of a correct base pair by a polymerase into a growing strand. However, this method is limited by the requirement of separate sequential addition of four individual nucleotides to determine the identity of the next nucleotide. Using ISFET detection, samples can be distributed on an array at the sub-micron level, and multiple arrays can be read simultaneously in a single device.
What is needed therefore, are improved methods, compositions, substrates and arrays for determining a polynucleotide sequence based on electronic detection to allow reduce feature size on an array for increased information density with output that allows for more efficient analysis.
Furthermore, arrays comprising primers or probes to bind to target sequences to allow sequencing are also needed to enable efficient binding of target polynucleotides for to an array for subsequent sequencing. Also needed are methods and compositions for manufacturing arrays comprising the probes.